PROJECT SUMMARY The combination antibiotic trimethoprim-sulfamethoxazole (TMP-SMX) is effective, inexpensive, and widely prescribed, yet it also causes idiosyncratic adverse drug reactions (IADRs) in 3-5% of TMP-SMX exposed patients, a rate much higher than that of most other drugs. Particularly alarming is that these IADRs are occurring with increasing frequency subsequent to increasing use. IADRs are feared by prescribers because they can result in unpredictable morbidity and even mortality. Despite these problems, TMP-SMX remains a critical mainstay therapy for treating infections worldwide because there are few widely available alternatives. Therefore it is essential to increase our mechanistic understanding of TMP-SMX IADRs in order to develop clinically relevant biomarkers that can predict risk. Although TMP-SMX is a combination antibiotic made up of two individual drug components, IADR mechanistic studies have to date been limited to SMX despite considerable clinical evidence that TMP contributes to these undesired reactions. The exact mechanism that leads to TMP-SMX IADRs has yet to be elucidated; however, bioactivation of a parent drug to a chemically reactive metabolite that covalently binds to a protein is considered to be an essential initiating event for IADR development. Recent data shows that TMP is bioactivated to reactive metabolites that can covalently bind to protein. TMP adducts have been identified in the urine of children taking TMP-SMX, suggesting that reactive TMP intermediates are formed in vivo. Currently it is not known whether patients experiencing TMP-SMX IADRs are responding to SMX, or to TMP, or to both, making it impossible to identify prospective patients who might be susceptible to an IADR caused by either component of TMP-SMX. We hypothesize that TMP may be a significant contributor to IADRs observed in TMP-SMX exposed patients. We will address this hypothesis in the following specific aims: 1) determine whether individuals treated with TMP- SMX have TMP and/or SMX protein adducts in their circulation 2) determine immunologic markers associated with TMP-SMX IADRs as related to each individual drug component and 3) determine if TMP metabolites are capable of covalently modifying proteins in the skin. Our long-term goal is to increase the clinical safety profile of this essential antibiotic. This research will contribute critical information about the mechanism behind TMP-SMX IADR development. Future studies will be directed in developing a tool that could be applied clinically to determine risk and enhance safe prescribe of TMP-SMX.